bioRxiv preprint doi: https://doi.org/10.1101/088229; this version posted November 17, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. miR-106b-responsive gene landscape identifies regulation of Kruppel- like factor family Cody J. Wehrkamp Sathish Kumar Natarajan Ashley M. Mohr Mary Anne Phillippi Justin L. Mott Department of Biochemistry and Molecular Biology, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha Running title: miR-106b-responsive genes in cholangiocarcinoma Key words: Apoptosis, biliary tract cancer, KLF2, KLF4, KLF6, KLF10, KLF11, KLF13, LKLF, lung Kruppel-like factor, miR-106a, miRNA, next-generation sequencing Address for Correspondence: Justin L. Mott, MD, PhD Associate Professor Department of Biochemistry and Molecular Biology 985870 Nebraska Medical Center Omaha, NE 68198-5870 Tel: 402-559-3177 Fax: 402-559-6650 e-mail: [email protected] List of abbreviations: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT; 4′,6-diamidine-2′- phenylindole dihydrochloride, DAPI; Crosslinking, ligation, and sequencing of hybrids, CLASH; Death receptor-5, DR5; Dulbecco’s Modified Eagle’s Medium, DMEM; False discovery rate, FDR; Fetal bovine serum, FBS; Kruppel-like factor, KLF; Locked-nucleic acid, LNA; Quantitative reverse-transcription PCR, qRT-PCR; Sodium dodecylsulfate-polyacrylamide gel electrophoresis, SDS-PAGE; Untranslated region, UTR; bioRxiv preprint doi: https://doi.org/10.1101/088229; this version posted November 17, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. miR-106b-responsive gene landscape identifies regulation of Kruppel-like factor family Abstract: MicroRNA dysregulation is a common feature of cancer and due to the promiscuity of microRNA binding this can result in a wide array of genes whose expression is altered. miR- 106b is an oncomiR overexpressed in cholangiocarcinoma and its upregulation in this and other cancers often leads to repression of anti-tumorigenic targets. The goal of this study was to identify the miR-106b-regulated gene landscape in cholangiocarcinoma cells using a genome- wide, unbiased mRNA analysis. Through RNA-Seq we found 112 mRNAs significantly repressed by miR-106b. The majority of these genes contain the specific miR-106b seed- binding site. We have validated 11 genes from this set at the mRNA level and demonstrated regulation by miR-106b of five proteins. Combined analysis of our miR-106b-regulated mRNA data set plus published reports indicate that miR-106b binding is anchored by G:C pairing in and near the seed. Novel targets Kruppel-like factor 2 (KLF2) and KLF6 were verified both at the mRNA and at the protein level. Further investigation showed regulation of four other KLF family members by miR-106b. We have discovered coordinated repression of several members of the KLF family by miR-106b that may play a role in cholangiocarcinoma tumor biology. Introduction microRNA which is vital for complementary binding to its mRNA target. The miR-106b has been established as an complementary seed-binding site is more or oncogenic microRNA with increased less conserved among targets, allowing for expression in many cancers including broad transcript-expression dampening cholangiocarcinoma [1-5], prostate [6], effects from a single microRNA. Seed- gastric [7], and hepatocellular carcinoma [8]. binding sites are highly enriched in the 3’ Some functions of miR-106b include untranslated region (UTR) of transcripts increased proliferation by miR-106b- [17]. There is also evidence that supports mediated reduction of the transcription non-canonical, non-seed mRNA target factor E2F1 [9] and the tumor suppressor interactions between microRNAs and their RB1 [10]. Additionally, miR-106b increased targets [18, 19]. The traditional seed for migration through targeting of the miR-106b is located at nucleotides 1-8 on phosphatase PTEN [11], and reduced its 5’ end, corresponding to the microRNA apoptosis by regulating the BH3-containing sequence 5’UAAAGUGC. protein Bim [12]. In this study, we sought to define the MicroRNAs function to dampen the genome-wide target set of miR-106b and expression of their targets [13-15]. This is found that miR-106b binding relied on accomplished both through reduction in sequences between bases 2-10, tolerating a mRNA transcript level as well as by G:U wobble in the seed region. We found repression of translation. While both 112 transcripts that were regulated at the mechanisms contribute to reduced target mRNA level in cholangiocarcinoma cells. protein expression, the effect of mRNA Finally, we demonstrated that members of reduction may dominate [16], in part the KLF family of transcription factors are because each message is used as a coordinately repressed by miR-106b. transcript for synthesis of many protein Comparison of our data in molecules; still, the magnitude of change in cholangiocarcinoma cells with data in Flp-In mRNA expression is low. A focused T-REx 293 human embryonic kidney- description of the binding characteristics derived cells [19] revealed that the and targets of individual microRNAs is microRNA-responsive gene set is largely feasible and desirable. cell-type specific. The seed region of a microRNA is a 7-8 nucleotide sequence at the 5’ end of the bioRxiv preprint doi: https://doi.org/10.1101/088229; this version posted November 17, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Materials and Methods Sequences were analyzed by the Bioinformatics and Systems Biology Core at Cell lines: Human cholangiocarcinoma cell the University of Nebraska Medical Center lines were previously derived from a female (UNMC) using the Tuxedo protocol [23]. patient with metastatic gallbladder cancer, Reads were mapped to the human genome Mz-ChA-1 [20], or a male patient with using Top Hat. Transcripts were assigned combined histologic features of by Cuff links and the transcriptome defined hepatocellular carcinoma and using Cuffmerge. Finally, differential cholangiocarcinoma, KMCH [21]. BDEneu expression between miR-106b and LNA- rat cholangiocarcinoma cells were derived 106b was calculated with Cuffdiff. from primary Fisher 344 rat cholangiocytes [22]. H69 cells are a non-malignant Using a false discovery rate (FDR)- immortalized cholangiocyte cell line. corrected p value, genome-wide transcripts Cholangiocarcinoma cells were grown in were ranked from most significant to least. Dulbecco’s Modified Eagle’s Medium This ranked list of genes was submitted to (DMEM) supplemented with 10% fetal the SylArray online server to detect bovine serum (FBS), insulin (0.5 µg/mL) enrichment of microRNA seed-binding sites and G418 (50 µg/mL). H69 cells were [24]. grown in DMEM supplemented with 10% miR-106b binding site analysis: RefSeq FBS, insulin (5 µg/mL), adenine (24.3 sequences for the 129 significant genes µg/mL), epinephrine (1 ug/mL), tri- (112 decreased and 17 increased) were iodothyronine (T3)- transferrin (T), [T3- 2.23 collected in Fasta format and analyzed by ng/ml, T-8.19 µg/mL], epidermal growth direct search for the known miR-106b factor (9.9 ng/mL) and hydrocortisone (5.34 binding site as well as subjected to k-mer µg/mL). analysis to generate a k table of all possible Cells were transfected either with mature 7-mer and 8-mer sequences and their miR-106b mimic (Life Technologies), frequency in this gene set. Several genes locked-nucleic acid (LNA) antagonist to had more than one RefSeq sequence, so miR-106b, LNA-106b (Exiqon), or LNA the final size of this Fasta file was 191 Negative Control A (Exiqon) for 24-48 hours transcripts. For comparison, 1,000 random using Lipofectamine RNAiMAX at a final sets of 191 transcripts each from the oligonucleotide concentration of 20 nM. RefSeq database were generated. miR-106b levels were quantified by TaqMan RNAHybrid [25] was used to identify the Small RNA assay (Life Technologies). single best miR-106b predicted binding site RNA-Seq: High quality total RNA was for each decreased transcript based on extracted from Mz-ChA-1 cells transfected minimum free energy hybridization. The with either miR-106b (n=3) or LNA-106b resulting miR-106b-binding sites were (n=3) for RNA-Seq using RNeasy Mini Kit manually curated for discovery of ungapped (Qiagen). The quality of the RNA was motifs using MEME Suite [26]. confirmed by RNA Integrity Analysis using Quantitative reverse-transcription PCR: an Agilent Bioanalyzer. RNA sequencing Total RNA for qRT-PCR was isolated using libraries were prepared by the UNMC Next TRIzol Reagent (Life Technologies) and Generation Sequencing Core Facility quantified using SYBR Green DNA binding beginning with 1 µg of total RNA and a (Roche). Primer pairs are listed in Table 1. TruSeq RNA Prep V2 (Illumina Inc, San Diego, CA). Samples were sequenced MicroRNA biotinylation: Biotinylation of Cel- using the Illumina HiSeq 2000 system with miR-67 and miR-106b was performed as 100 bp paired-end reads. An average of described [27]. Mature microRNA for C. 76.99 million reads per sample were elegans miR-67 (control) and H. sapiens collected (range 58.99-87.00 million). miR-106b (both leading and passenger bioRxiv preprint doi: https://doi.org/10.1101/088229; this version posted November 17, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.